The ability to select a small number of cells from a heterogeneous population is fundamental to many aspects of biological research. Selections form the basis of genetic screens, of protein engineering and directed evolution, and of protocols to produce stably transformed or genome-edited cell lines. In many instances, one would like to select cells on the basis of complex dynamic or morphological features. For example, in a culture of olfactory neurons, one might screen for calcium flux in response to a specific odorant and then wish to select responsive cells for subsequent transcriptional profiling. Or in a culture with single genes knocked down by an siRNA library,1, 2 one might find cells with unusual shapes, organelle sizes, or metabolic responses; and then wish to select these cells to determine which gene had been knocked down. These types of selections are difficult to perform with existing tools.
Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g., a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g., fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection).
The most common selection technique uses fluorescence-activated cell sorting (FACS),3 which requires a robust static fluorescence signal. In FACS, cells are suspended in a fine stream of droplets which pass through one or more laser detection points. Cells whose fluorescence falls within user-specified bounds are electrostatically deflected into a collection well.3 There are several limitations to this method. FACS requires a robust fluorescence signal. Due to the limited observation time per cell (typically about 10 μs) the level of noise is high, and it is not possible to discriminate small changes in fluorescence levels or weak fluorescence signals. FACS probes fluorescence at only a single moment in time. Dynamical quantities, e.g., beat rate, locomotion, subcellular transport, or timecourse of response to a perturbation are not amenable to FACS. FACS also does not provide structural information. Morphological attributes, such as cell shape or subcellular distribution of mitochondria are not amenable to FACS. FACS requires cells to be in suspension. Thus parameters that are disrupted by suspending cells are not a suitable basis for FACS. Some cells such as neurons suffer damage or low viability when suspended in solution.
Laser-capture microdissection (LCM)4, 5 selects cells or tissue regions one at a time, and so can have limited throughput, and is usually performed on samples that have been chemically fixed. LCM selects cells or tissue regions with a brief and intense pulse of laser light which catapults the selected cells onto a capture membrane.4, 5 In some variations on LCM, a thermo-adhesive membrane is locally heated by a laser and thereby locally adheres to the cells of interest. The limitations of LCM are: due to its serial nature, LCM can have limited throughput; LCM is usually performed on samples that have been chemically fixed, and so is not readily compatible with dynamical properties of cells, nor with subsequent cell growth; LCM requires expensive and highly specialized instrumentation.
Imaging cytometry6, 7 typically functions in a flow-through geometry, and so is not compatible with selections of surface-bound cells such as neurons; nor with selections that probe dynamic cellular responses.
Photochemical selection techniques such as spatially patterned photodegradation of the cell culture substrate has been demonstrated as a means for selecting cells from culture.16, 27 In principle this approach allows for the selection of cells on the basis of complex criteria. Spatially patterned photochemistry is becoming widely applied in cell biology for its ability to induce specific reactions in complex patterns of space and time.8 Photochemical pre-patterning of cell adhesion molecules enables cell growth in complex morphologies,9-11 and photopatterned hydrogels are now used to direct cell culture in three dimensions.12-14 In these applications the pattern is defined prior to plating the cells. For screening purposes one would like to define the adhesion pattern after plating the cells, only retaining cells with a user-specified phenotype. Two recent demonstrations showed photochemical release of cells from a photodegradable surface,15, 16 but in these protocols the surface had to be specially prepared prior to cell culture. The technique has the following limitations. When cells are in contact, cell-cell bonds can prevent isolation of a single cell simply by disrupting the substrate. The techniques require the cells to be cultured on a specially prepared substrate. Long-term culture on a photodegradable substrate may affect cell viability, is not compatible with imaging modalities that would degrade the substrate, and may not be compatible with some cell culture protocols.
Therefore, there remains a need for systems and methods for cell selection from heterogeneous cell cultures while also preserving cell viability.